This invention relates to a method for preparing .beta.-lactam-type antibiotic test discs used in a clinical microbiological test procedure.
More specifically, this invention concerns a method for preparing .beta.-lactam-type-antibiotic susceptibility test discs which are significantly more stable to heat and storage than those test discs heretofore available from prior art processes. Such enhanced stability greatly increases the reliability of the microbiological test results obtained when such discs are used to determine the susceptibility of pathological microorganisms to the .beta.-lactam-type-antibiotic contained on such discs.
Antibiotic susceptibility test discs have been used for a number of years to determine qualitatively the susceptibility of various pathological microorganisms to the antibiotic contained on such discs. Bauer A. W., et al. [Bauer A. W., Kirby, W. M. M., Sherris, J. C., and Turch, M., Antibiotic Susceptibility Testing by a Standardized Single Disc Method, Am J. Chem Path., 45, 493, (1966)] described the test procedure wherein such test discs are utilized. Usually, the tests are run in hospital laboratories where facilities are available to run microbiological tests. There are, however, some medical laboratories which are independent of hospitals which have the capability to run such tests.
Antibiotic susceptibility tests are run by inoculating a sterile nutrient agar medium, such as Mueller-Hinton agar, with a properly diluted bacterial suspension prepared from a bacterial isolate. The preparation of the inocula and the inoculation procedure are well known to those skilled in the art. The inoculated agar is allowed to dry for 15 to 20 minutes and an antibiotic susceptibility test disc having a specified amount of antibiotic contained thereon, 30 mcg in the case of cephalothin and 10 international units for penicillin G, is placed on the inoculated agar and gently pressed down with a sterile instrument to assure a uniform contact. The thus prepared sample is incubated at 37.degree.C. for 24 hours. An indication of the susceptibility of the pathological microorganism to the antibiotic contained on the test disc is obtained by observing and measuring the diameter of the surface of the medium surrounding the test disc that is free of microorganism growth. For example, if the inhibition zone surrounding a test disc containing 30 mcg of cephalothin is 14 mm or less in diameter, it is indicated that the microorganism is resistant to cephalothin. If the zone diameter is 18 mm or more, the microorganism is judged to be susceptible to cephalothin. Standards have also been established for other antibiotics which are tested in the same manner. These can be found in the Federal Register.
The susceptibility discs are of clean, white paper and can be of different diameters, one popular size being one quarter inch (6.35 mm) in diameter. The standards call for the paper to weigh 30.+-.4 mg per cm.sup.2 and to have an absorbtive capacity for distilled water of about 2-3 times the weight of the paper. The paper shall contain no material which will enhance or inhibit the activity of the antibiotic added to it, and can have no effect on the pH of the agar medium or the antibiotic. One grade of paper that meets all of the specifications is alpha cellulose paper No. 676 which can be obtained from Eaton-Dikeman Company, Mount Holly Springs, Pennsylvania.
In the present commercial process, sheets of paper meeting the above detailed specifications are dipped into a solution of the antibiotic, saturated with such solution and removed therefrom. The saturated sheets are subjected to a mechanical compression to remove a substantial portion of the antibiotic solution leaving an amount of solution calculated to deposit the requisite quantity of antibiotic on each one-quarter inch diameter segment of such paper after the solvent is evaporated therefrom. The compressed sheets are then dried at an appropriate temperature, for example, 90.degree.C. in the case of cephalothin, to remove the solvent therefrom and one-quarter inch diameter discs are die cut from the dried paper.
The procedure outlined above results in the deposit of dry amorphous antibiotic in the amount desired on each disc; 30 mcg in the case of cephalothin.
It was found that .beta.-lactam-type-antibiotic susceptibility test discs prepared by this prior art process are highly unstable to heat and storage, losing as much as two-thirds or more of the antibiotic activity after 90 days storage at 37.degree.C.; over half of which was lost in the first thirty days of the storage. Consequently, in order to assure the stated potency, such discs must be refrigerated in actual hospital practice.
Accordingly, it is an object of this invention to provide .beta.-lactam-type-antibiotic susceptibility test discs that will retain substantially all of the antibiotic activity without requiring refrigeration and, in addition, exhibit significantly improved stability on continued storage at 37.degree.C. for up to 90 days over those susceptibility test discs prepared by the prior art process.
Another object of this invention is to provide a method for preparing antibiotic susceptibility test discs having good stability to heat and storage in that such discs retain substantially all of the antibiotic activity when stored at 37.degree.C. for 30 days.